Inhibition of SARS-associated coronavirus (SCoV) infection and replication by RNA interference

ABSTRACT

The present invention relates to therapeutic agents useful for the treatment of Severe Acute Respiratory Syndrome (SARS) in humans. In particular, the present invention relates to RNA interference (RNAi) molecules useful for inhibiting the infection and replication of hSARS virus. Preferably, the RNAi molecules target the replicase region of the hSARS virus, or combinations of different sites of hSARS virus genes. The present invention further encompasses methods of using the RNAi molecules for preventing and/or treating SARS. Vaccines and kits comprising therapeutically effective amounts of the RNAi molecules are also encompassed.

This application is a division of U.S. patent application Ser. No.10/848,737, filed May 19, 2004, now U.S. Pat. No. 7,129,223, issued Oct.31, 2006; which claims the benefit of U.S. provisional application No.60/471,901, filed May 19, 2003, both of which are incorporated herein byreference in their entireties.

1. INTRODUCTION

The present invention relates to therapeutic agents useful for thetreatment of Severe Acute Respiratory Syndrome (SARS) in humans. Theetiologic agent of SARS is a human RNA virus known as the hSARS virus.The hSARS virus is identified to be morphologically and phylogeneticallysimilar to known members of Coronaviridae. The present invention relatesto nucleic acid molecules comprising a nucleotide sequence which wasdesigned based on portions of the genomic sequences of the differentstrains of the hSARS virus and their use as therapeutic agents intherapeutic methods. Specifically, the present invention relates to genetherapy, or the use of RNA interference (RNAi) molecules such asdouble-stranded RNA (dsRNA) or small interference RNA (siRNA) astherapeutic agents for the treatment of SARS in humans. Preferably, theRNAi molecules target the replicase region of the hSARS virus.

2. BACKGROUND OF THE INVENTION

Recently, there has been an outbreak of atypical pneumonia in Guangdongprovince in mainland China. Between November 2002 and March 2003, therewere 792 reported cases with 31 fatalities (WHO. Severe AcuteRespiratory Syndrome (SARS) Weekly Epidemiol Rec. 2003; 78: 86). Inresponse to this crisis, the Hospital Authority in Hong Kong hasincreased the surveillance on patients with severe atypical pneumonia.In the course of this investigation, a number of clusters of health careworkers with the disease were identified. In addition, there wereclusters of pneumonia incidents among persons in close contact withthose infected. The disease was unusual in its severity and itsprogression in spite of the antibiotic treatment typical for thebacterial pathogens that are known to be commonly associated withatypical pneumonia. The disease was given the acronym Severe AcuteRespiratory Syndrome (“SARS”).

A novel coronavirus associated with SARS (herein interchangeablyreferred to “SCoV”, “CoV” or “hSARS” virus) has been identified as theetiologic agent of SARS (Ksiazek, T G, et al. A Novel CoronavirusAssociated with Severe Acute Respiratory Syndrome. N. Engl. J. Med.2003; published online (10.1056/NEJMMoa030781); Marra, M A, et al. TheGenome Sequence of the SARS-Associated Coronavirus. Science 2003;published online (10.1126/science.1085953); Peiris, J S, et al.Coronavirus as a possible cause of severe acute respiratory syndrome.Lancet 2003; 361: 1319-1325; Drosten, C, et al. Identification of aNovel Coronavirus in Patients with Severe Acute Respiratory Syndrome. NEngl J Med. 2003; published online (10.1056/NEJMoa030747); and Rota, PA, et al. Characterization of a Novel Coronavirus Associated with SevereAcute Respiratory Syndrome. Science 2003; (10.1126/science.1085952)).The complete genome sequences of coronavirus strains isolated fromdifferent patients in various geographic locations were reportedrecently (Marra et al., supra.; Rota et al., supra.; and U.S. patentapplication Ser. Nos. 60/464,886, filed Apr. 23, 2003, and 10/808,121filed Mar. 24, 2004; each of which is incorporated herein by referencein its entirety). The isolated virus is an enveloped, single-strandedRNA virus of positive polarity which belongs to the order, Nidovirales,of the family, Coronaviridae. The total genome of the SARS coronavirusis 29,727 nucleotides in length (see, for example, Genbank NCBIAccession Nos: AY274119, AY304495 and AY278491). This is the largestgenome yet found in any of the known RNA viruses. The genomeorganization of the hSARS virus is similar to that of othercoronaviruses. It encodes for at least 5 gene products, i.e., replicase,spike glycoprotein (S), membrane protein (M), envelope protein (E) andnucleocapsid protein (N), with eleven possible open reading frames (seeFIGS. 1A and 4A). The exact numbers of gene products are not clear yet.Currently, there is no known effective treatment for SARS. Accordingly,drug development for the treatment of SARS is urgently needed. Theinventors have formulated RNAi molecules useful for inhibiting theinfection and replication of the hSARS virus. This offers thepossibility of developing a new anti-viral therapy for SARS.

3. SUMMARY OF INVENTION

The present invention is based upon the inventor's use of gene therapysuch as RNA interference (RNAi) molecules to inhibit coronavirusinfection and replication. In particular, the invention relates to theuse of small interfering RNA (siRNA) or double-stranded RNA (dsRNA) astherapeutic agents for the treatment of Severe Acute RespiratorySyndrome (SARS) in humans. The invention encompasses RNAi molecules thattarget different sites of the genome of the hSARS virus and inhibitinfection and replication of the hSARS virus. Specifically, theinvention encompasses siRNAs that target different sites of thereplicase region of the hSARS virus and are useful for the treatment ofSARS in humans. The present invention also encompasses small hairpin RNA(shRNA) containing plasmids under the control of a promoter useful forthe treatment of SARS.

In certain embodiments, the invention relates to nucleic acid moleculescomprising a portion of the genomic sequence of the hSARS virus. Inpreferred embodiments, the invention relates to nucleic acid moleculesisolated from a replicase region of the genomic sequence of the hSARSvirus. In certain other embodiments, the invention relates to nucleicacid molecules isolated from the spike glycoprotein (S) region, themembrane protein (M) region, the envelope protein (E) region, and thenucleocapsid protein (N) region of the hSARS viral genome.

In a specific embodiment, the nucleic acid molecules encode apolypeptide comprising a portion of the hSARS virus. Preferably, thenucleic acid molecules encode a polypeptide, or a portion thereof,comprising the replicase region of the hSARS virus.

In a specific embodiment, the invention relates to nucleic acidmolecules encoding a polypeptide comprising a portion of the genomicsequence of the replicase region of the hSARS virus, or a portionthereof. Preferably, the replicase region of the hSARS virus is one thatis described in FIG. 1 and Section 5, infra. In a preferred embodiment,the nucleic acid molecule comprises the nucleotide sequence of SEQ IDNO:1, 2, 3, 4, 5 or 6. In a specific embodiment, the present inventionprovides isolated nucleic acid molecules comprising or, alternatively,consisting of the nucleotide sequence of SEQ ID NO:1, a complementthereof or a portion thereof, preferably at least 5, 10, 15, 20, or morecontiguous nucleotides of the nucleotide sequence of SEQ ID NO:1, or acomplement thereof. In another specific embodiment, the presentinvention provides isolated nucleic acid molecules comprising or,alternatively, consisting of the nucleotide sequence of SEQ ID NO:2, acomplement thereof or a portion thereof, preferably at least 5, 10, 15,20, or more contiguous nucleotides of the nucleotide sequence of SEQ IDNO:2, or a complement thereof. In yet another specific embodiment, thepresent invention provides isolated nucleic acid molecules comprisingor, alternatively, consisting of the nucleotide sequence of SEQ ID NO:3,a complement thereof or a portion thereof, preferably at least 5, 10,15, 20, or more contiguous nucleotides of the nucleotide sequence of SEQID NO:3, or a complement thereof. In yet another specific embodiment,the present invention provides isolated nucleic acid moleculescomprising or, alternatively, consisting of the nucleotide sequence ofSEQ ID NO:4, a complement thereof or a portion thereof, preferably atleast 5, 10, 15, 20, or more contiguous nucleotides of the nucleotidesequence of SEQ ID NO:4, or a complement thereof. In yet anotherspecific embodiment, the present invention provides isolated nucleicacid molecules comprising or, alternatively, consisting of thenucleotide sequence of SEQ ID NO:5, a complement thereof or a portionthereof, preferably at least 5, 10, 15, 20, or more contiguousnucleotides of the nucleotide sequence of SEQ ID NO:5, or a complementthereof. In yet another specific embodiment, the present inventionprovides isolated nucleic acid molecules comprising or, alternatively,consisting of the nucleotide sequence of SEQ ID NO:6, a complementthereof or a portion thereof, preferably at least 5, 10, 15, 20, or morecontiguous nucleotides of the nucleotide sequence of SEQ ID NO:6, or acomplement thereof.

In yet another embodiment, the invention relates to nucleic acidmolecules encoding a polypeptide comprising a portion of the genomicsequence of the spike glycoprotein (S) region, membrane protein (M)region, envelope protein (E) region, and/or nucleocapsid protein (N)region of the hSARS virus as described in FIG. 4A and Section 5, infra.In preferred embodiments, the nucleic acid molecule comprises thenucleotide sequence of SEQ ID NO:7, 8, 9, 10 or 11. In a specificembodiment, the present invention provides isolated nucleic acidmolecules comprising or, alternatively, consisting of the nucleotidesequence of SEQ ID NO:7, a complement thereof or a portion thereof,preferably at least 5, 10, 15, 20, or more contiguous nucleotides of thenucleotide sequence of SEQ ID NO:7, or a complement thereof. In anotherspecific embodiment, the present invention provides isolated nucleicacid molecules comprising or, alternatively, consisting of thenucleotide sequence of SEQ ID NO:8, a complement thereof or a portionthereof, preferably at least 5, 10, 15, 20, or more contiguousnucleotides of the nucleotide sequence of SEQ ID NO:8, or a complementthereof. In yet another specific embodiment, the present inventionprovides isolated nucleic acid molecules comprising or, alternatively,consisting of the nucleotide sequence of SEQ ID NO:9, a complementthereof or a portion thereof, preferably at least 5, 10, 15, 20, or morecontiguous nucleotides of the nucleotide sequence of SEQ ID NO:9, or acomplement thereof. In yet another specific embodiment, the presentinvention provides isolated nucleic acid molecules comprising or,alternatively, consisting of the nucleotide sequence of SEQ ID NO:10, acomplement thereof or a portion thereof, preferably at least 5, 10, 15,20, or more contiguous nucleotides of the nucleotide sequence of SEQ IDNO:10, or a complement thereof. In yet another specific embodiment, thepresent invention provides isolated nucleic acid molecules comprisingor, alternatively, consisting of the nucleotide sequence of SEQ IDNO:11, a complement thereof or a portion thereof, preferably at least 5,10, 15, 20, or more contiguous nucleotides of the nucleotide sequence ofSEQ ID NO:11, or a complement thereof. Methods of RNA interference arealso provided.

Furthermore, in another specific embodiment, the invention providesnucleic acid molecules which hybridize under stringent conditions, asdefined herein, to a nucleic acid molecule having the sequence of SEQ IDNO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, or a fragment thereof, or acomplement thereof. In one embodiment, the invention provides anisolated nucleic acid molecule which is antisense to the coding strandof a nucleic acid molecule of the invention.

In a specific embodiment, the invention provides isolated polypeptidesor proteins that are encoded by a nucleic acid molecule comprising or,alternatively consisting of a nucleotide sequence that is at least 5,10, 15, 20, or more contiguous nucleotides of the nucleotide sequence ofSEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, or a complement thereof.

The invention further provides antibodies that specifically bind apolypeptide of the invention encoded by the nucleotide sequence of SEQID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, or a fragment thereof, orencoded by a nucleic acid comprising a nucleotide sequence thathybridizes under stringent conditions to the nucleotide sequence of SEQID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, having one or more biologicalactivities of a polypeptide of the invention. Such antibodies include,but are not limited to polyclonal, monoclonal, bi-specific,multi-specific, human, humanized, chimeric antibodies, single chainantibodies, Fab fragments, F(ab′)2 fragments, disulfide-linked Fvs,intrabodies and fragments containing either a VL or VH domain or even acomplementary determining region (CDR) that specifically binds to apolypeptide of the invention.

The invention further relates to the use of the nucleic acid moleculesfor therapeutic methods. In a specific embodiment, the inventionprovides nucleic acid molecules which are suitable for administeringinto a subject in need thereof. In a preferred embodiment, the nucleicacid molecules comprise the nucleotide sequence of SEQ ID NO:1, 2, 3, 4,5, 6, 7, 8, 9, 10 or 11, or a complement thereof, or at least a portionof the nucleotide sequence thereof. In one embodiment, the nucleic acidmolecules are directly delivered into a host genome. In anotherembodiment, the nucleic acid molecules are encapsulated into liposomes.In yet another embodiment, the nucleic acid molecules are deliveredusing a vector system such as adenovirus, adeno-associated virus (AAV),or retrovirus.

In one embodiment, the invention provides methods for detecting thepresence, activity or expression of the hSARS virus of the invention ina biological material, such as cells, blood, saliva, urine,nasopharyngeal aspirates, feces, and so forth. The increased ordecreased activity or expression of the hSARS virus in a sample relativeto a control sample can be determined by contacting the biologicalmaterial with an agent which can detect directly or indirectly thepresence, activity or expression of the hSARS virus. In a specificembodiment, the detecting agents are the antibodies or nucleic acidmolecules of the present invention. Antibodies of the invention may alsobe used to treat SARS.

In another embodiment, the invention provides vaccine preparations,comprising the nucleic acid molecules of the present invention,including free and encapsulated forms of said nucleic acid molecule, orsubunits of the nucleic acid molecule. In another embodiment, theinvention provides vaccine preparations comprising one or morepolypeptides of the invention encoded by the nucleotide sequence of SEQID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, or a fragment thereof.Furthermore, the present invention provides methods for treating,ameliorating, managing or preventing SARS by administering the vaccinepreparations, antibodies or other anti-viral agents of the presentinvention alone or in combination with adjuvants, or otherpharmaceutically acceptable excipients.

In another aspect, the present invention provides pharmaceuticalcompositions comprising anti-viral agents of the present invention and apharmaceutically acceptable carrier. In a specific embodiment, theanti-viral agent of the invention is a nucleic acid molecule or thepolypeptide encoded by the nucleic acid molecule of the invention, orthe antibodies of the invention that immunospecifically binds hSARSvirus or any hSARS epitope. In another specific embodiment, theanti-viral agent is a nucleic acid molecule which hybridizes genome orother RNA species of the hSARS virus under physiological condition. Inanother specific embodiment, the anti-viral agent is a polypeptide orprotein of the present invention or nucleic acid molecule of theinvention. The invention also provides kits containing a pharmaceuticalcomposition of the present invention.

3.1 Definitions

The term “an antibody or an antibody fragment that immunospecificallybinds a polypeptide of the invention” as used herein refers to anantibody or a fragment thereof that immunospecifically binds to thepolypeptide encoded by the nucleotide sequence of SEQ ID NO:1, 2, 3, 4,5, 6, 7, 8, 9, 10 or 11, or a fragment thereof, and does notnon-specifically bind to other polypeptides. An antibody or a fragmentthereof that immunospecifically binds to the polypeptide of theinvention may cross-react with other antigens. Preferably, an antibodyor a fragment thereof that immunospecifically binds to a polypeptide ofthe invention does not cross-react with other antigens. An antibody or afragment thereof that immunospecifically binds to the polypeptide of theinvention, can be identified by, for example, immunoassays or othertechniques known to those skilled in the art.

An “isolated” or “purified” peptide or protein is substantially free ofcellular material or other contaminating proteins from the cell ortissue source from which the protein is derived, or substantially freeof chemical precursors or other chemicals when chemically synthesized.The language “substantially free of cellular material” includespreparations of a polypeptide/protein in which the polypeptide/proteinis separated from cellular components of the cells from which it isisolated or recombinantly produced. Thus, a polypeptide/protein that issubstantially free of cellular material includes preparations of thepolypeptide/protein having less than about 30%, 20%, 10%, 5%, 2.5%, or1%, (by dry weight) of contaminating protein. When thepolypeptide/protein is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, 10%, or 5% of the volume of the proteinpreparation. When polypeptide/protein is produced by chemical synthesis,it is preferably substantially free of chemical precursors or otherchemicals, i.e., it is separated from chemical precursors or otherchemicals which are involved in the synthesis of the protein.Accordingly, such preparations of the polypeptide/protein have less thanabout 30%, 20%, 10%, 5% (by dry weight) of chemical precursors orcompounds other than polypeptide/protein fragment of interest. In apreferred embodiment of the present invention, polypeptides/proteins areisolated or purified.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule or an RNA molecule, can be substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. In a preferred embodiment of theinvention, nucleic acid molecules encoding polypeptides/proteins of theinvention are isolated or purified. The term “isolated” nucleic acidmolecule does not include a nucleic acid that is a member of a librarythat has not been purified away from other library clones containingother nucleic acid molecules.

The term “portion” or “fragment” as used herein refers to a fragment ofa nucleic acid molecule containing at least about 5, 10, 15, 20, 25, 30,35, 40, 45, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000,11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000,20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000,29,000, or more contiguous nucleic acids in length of the relevantnucleic acid molecule and having at least one functional feature of thenucleic acid molecule (or the encoded protein has one functional featureof the protein encoded by the nucleic acid molecule); or a fragment of aprotein or a polypeptide containing at least 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 120, 140, 160, 180, 200,220, 240, 260, 280, 300, 320, 340, 360, 400, 420, 440, 460, 480, 500,520 or 540 amino acid residues in length of the relevant protein orpolypeptide and having at least one functional feature of the protein orpolypeptide.

The term “having a biological activity of the protein” or “havingbiological activities of the polypeptides of the invention” refers tothe characteristics of the polypeptides or proteins having a commonbiological activity similar or identical structural domain and/or havingsufficient amino acid identity to the polypeptide encoded by thenucleotide sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.Such common biological activities of the polypeptides of the inventioninclude antigenicity and immunogenicity.

The term “under stringent condition” refers to hybridization and washingconditions under which nucleotide sequences having at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%identity to each other remain hybridized to each other. Suchhybridization conditions are described in, for example but not limitedto, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6.; Basic Methods in Molecular Biology, ElsevierScience Publishing Co., Inc., N.Y. (1986), pp. 75-78, and 84-87; andMolecular Cloning, Cold Spring Harbor Laboratory, N.Y. (1982), pp.387-389, and are well known to those skilled in the art. A preferred,non-limiting example of stringent hybridization conditions ishybridization in 6× sodium chloride/sodium citrate (SSC), 0.5% SDS atabout 68° C. followed by one or more washes (e.g., about 5-30 min each)in 2×SSC, 0.5% SDS at room temperature. Another preferred, non-limitingexample of stringent hybridization conditions is hybridization in 6×SSCat about 45° C. followed by one or more washes (e.g., about 5-30 mineach) in 0.2×SSC, 0.1% SDS at about 45-65° C.

The term “variant” as used herein refers either to a naturally occurringgenetic mutant of hSARS or a recombinantly prepared variation of hSARS.The term “variant” may also refers either to a naturally occurringvariation of a given peptide or a recombinantly prepared variation of agiven peptide or protein in which one or more amino acid residues havebeen modified by amino acid substitution, addition, or deletion.

4. DESCRIPTION OF THE FIGURES

FIG. 1A shows the open reading frame and functional domains of the hSARSvirus. FIG. 1B shows the sense-strand sequences of six 21- and 22-mersiRNAs that target different sites of the replicase 1A region: SARSi-1corresponds to the coronavirus nucleotide sequence 512 to 531 (SEQ IDNO:1); SARSi-2 corresponds to the coronavirus nucleotide sequence 586 to604 (SEQ ID NO:2); SARSi-3 corresponds to the coronavirus nucleotidesequence 916 to 934 (SEQ ID NO:3); SARSi-4 corresponds to thecoronavirus nucleotide sequence 1194 to 1213 (SEQ ID NO:4); SARSi-5corresponds to the coronavirus nucleotide sequence 3028 to 3046 (SEQ IDNO:5); and SARSi-6 corresponds to the coronavirus nucleotide sequence5024 to 5042 (SEQ ID NO:6).

FIG. 2 shows the morphological changes with cytopathic effect (CPE) andimmunostaining with antibody against coronavirus antigens of monkeykidney cells (FRhk-4 cells) infected with coronavirus (GZ50 strain).FRhk-4 cells were transfected with the siRNAs prior to infection withcoronavirus. (A) shows the morphology and (I) shows antibody staining ofuninfected cells. (B) shows the morphology and (J) shows antibodystaining of SCoV-infected cells. (C)-(H) show the morphology and (K)-(P)show antibody staining of infected cells transfected with the siRNAs:SARSi-1, SARSi-2, SARSi-3, SARSi-4, SARSi-5, and SARSi-6, respectively.

FIG. 3A shows the viral genomic RNA of infected cells with or withoutthe treatment with siRNAs as determined by reverse transcription andpolymerase chain reaction (RT-PCR). FIG. 3B shows the viral titer ofcells infected with three (3) strains of coronavirus: GZ34 strain, HKR1strain and HKR2 strain, respectively, with or without the treatment withSARSi-4.

FIG. 4A shows the physical map of SCoV. The targeting sites are shown byarrows. FIG. 4B shows the sense-strand sequences of five 21-, 22- and23-mer siRNAs that target one site of the S glycoprotein region(SARSi-7, corresponding to the coronavirus nucleotide sequence 23165 to23184; SEQ ID NO:7), one site of the E protein region (SARSi-8,corresponding to the coronavirus nucleotide sequence 26128 to 26148; SEQID NO:8), one site of the N protein region (SARSi-9, corresponding tothe coronavirus nucleotide sequence 28663 to 28682; SEQ ID NO:9), andtwo sites of the M protein region (SARSi-10, corresponding to thecoronavirus nucleotide sequence 26652 to 26671; SEQ ID NO:10, andSARSi-11, corresponding to the coronavirus nucleotide sequence 26575 to26595; SEQ ID NO:11), respectively.

FIG. 5 shows the cytopathic effect (“CPE”) on FRhk-4 cells. FRhk-4 cellswere infected with SCoV at multiplicity of infection (“MOI”) of 0.05(II, IV to X) with (IV-X) or without siRNA (II). Non-infected cell (I)and GL2i-transfected cell without SCoV infection (III) served ascontrols. The photos were taken under phase-contrast microscopy (400×)with a light filter 24 hours post-infection. The arrows show the sickcells. “Si-” denotes “SARSi-”.

FIGS. 6A-6D show inhibition of SCoV replication and reproduction bysiRNAs. FIG. 6A shows the levels of intracellular viral RNA copies atdifferent time points after infection. FRhk-4 cells were transfectedwith siRNAs and infected with SCoV. The cellular RNA was isolated atdifferent time points and quantitative RT-PCR was conducted. Theexperiments were performed in triplicate and repeated at least threetimes. The values (mean±standard error) represent the mean of threeindependent experiments. The viral genomic RNA copies per cell at 24hours were calculated: GL2i, 1.69×10⁶±4.7×10³; SARSi-4, 1.2×10⁵±5.5×10²;SARSi-7, 2.5×10⁵±1.0×10⁵; SARSi-8, 4.4×10⁵±1.0×10⁵; SARSi-9,3.8×10⁵±2.2×10⁵; SARSi-10, 3.1×10⁵±1.2×10⁵; and SARSi-11,5.7×10⁵±1.5×10⁵. FIG. 6B shows relative viral titers of SCoV in theculture media measured by back titration. The mean value of viral titersobtained from GL2i control was defined as 100. The relative viral titersof siRNAs targeted on SARS were: SARSi-4, 2.1±0.3; SARSi-7, 1.9±0.0.4;SARSi-8, 1.1±0.2; SARSi-9, 18.4±1.6; SARSi-10, 2±0.3; and SARSi-11,2.2±0.4. FIG. 6C shows that siRNAs inhibited SCoV replication in adose-dependent manner. FIG. 6D shows the effects of siRNAs used incombination. FRhk-4 cells were transfected with single siRNA (10 nM), ortwo combined siRNAs (5 nM each), and infected with SCoV (MOI of 0.05).At 24 hours post-infection the viral titers in the culture media weredetermined by back titration. The mean value of control (GL2i samples)was defined as 100. The values were as follows: GL2i, 100±12.8; Si-4,16.7±3.8; Si-7, 54.2±12.1; Si-8, 13.5±2.8; Si-9, 54.8±10.8; Si-10,50.2±13.2; Si-4/7, 5.6±1.3; Si-4/8, 8.3±1.9; Si-4/9, 17.3±4.2; Si-7/8,5.8±1.2; and Si-7/10, 16.5±3.4.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to RNA interference (RNAi) moleculesuseful for inhibiting coronavirus infection and replication. Inparticular, the invention relates to small interfering RNA (siRNA) anddouble-stranded RNA (dsRNA) useful for inhibiting coronavirus infectionand replication. The present invention relates to nucleic acid moleculescomprising portions of the genomic sequence of the hSARS virus,preferably the replicase region of the hSARS virus.

5.1 hSARS Virus

The present invention relates to the isolated hSARS virus thatmorphologically and phylogenetically relates to known Coronaviruses. Ina specific embodiment, the virus comprises a nucleotide sequence of SEQID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and/or 11. In a specific embodiment,the present invention provides isolated nucleic acid molecules of thehSARS virus, comprising, or, alternatively, consisting of the nucleotidesequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and/or 11, acomplement thereof or a portion thereof. In another specific embodiment,the invention provides isolated nucleic acid molecules which hybridizeunder stringent conditions, as defined herein, to a nucleic acidmolecule having the sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10or 11, or specific genes of known member of Coronaviridae, or acomplement thereof.

In yet another specific embodiment, the invention provides isolatedpolypeptides or proteins that are encoded by a nucleic acid moleculecomprising or, alternatively consisting of a nucleotide sequence that isat least 5, 10, 15, 20 or more contiguous nucleotides of the nucleotidesequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, or acomplement thereof. The polypeptides or the proteins of the presentinvention preferably have one or more biological activities of theproteins encoded by the sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9,10 or 11, or the native viral proteins containing the amino acidsequences encoded by the sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8,9, 10 and/or 11.

The invention further relates to the use of the sequence information ofthe isolated virus for and therapeutic methods. In a specificembodiment, the present invention relates to a nucleic acid moleculethat hybridizes any portion of the genome of various strains of thehSARS virus, including Genbank NCBI Accession Nos. AY304495, AY274119and AY278491, under the stringent conditions. In a specific embodiment,the invention provides nucleic acid molecules which are suitable for useas primers consisting of or comprising the nucleotide sequence of SEQ IDNO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and/or 11, or a complement thereof, ora portion thereof. In another specific embodiment, the inventionprovides nucleic acid molecules which are suitable for use ashybridization probes for the detection of nucleic acids encoding apolypeptide of the invention, consisting of or comprising the nucleotidesequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, a complementthereof, or a portion thereof. The invention further encompasseschimeric or recombinant viruses or viral proteins encoded by saidnucleotide sequences.

The invention further provides antibodies that immunospecifically bind apolypeptide of the invention encoded by the nucleotide sequence of SEQID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, or a fragment thereof, or anyhSARS epitope. Such antibodies include, but are not limited topolyclonal, monoclonal, bi-specific, multi-specific, human, humanized,chimeric antibodies, single chain antibodies, Fab fragments, F(ab′)₂fragments, disulfide-linked Fvs, intrabodies and fragments containingeither a VL or VH domain or even a complementary determining region(CDR) that specifically binds to a polypeptide of the invention.

In one embodiment, the invention provides methods for detecting thepresence, activity or expression of the hSARS virus of the invention ina biological material, such as cells, blood, saliva, urine, sputum,nasopharyngeal aspirates, and so forth. The presence of the hSARS virusin a sample can be determined by contacting the biological material withan agent which can detect directly or indirectly the presence of thehSARS virus. In a specific embodiment, the detection agents are theantibodies of the present invention. In another embodiment, thedetection agent is a nucleic acid of the present invention.

In another embodiment, the invention provides vaccine preparationscomprising one or more nucleic acid molecules comprising or consistingof the sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and/or 11, ora fragment thereof. In another embodiment, the invention providesvaccine preparations comprising one or more polypeptides of theinvention encoded by a nucleotide sequence comprising or consisting ofthe nucleotide sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10and/or 11, or a fragment thereof. The vaccine preparations of thepresent invention may further comprise pharmaceutically acceptableexcipients, including adjuvants.

Furthermore, the present invention provides methods for treating,ameliorating, managing, or preventing SARS by administering the RNAinterference (RNAi) molecules of the present invention alone or incombination with antivirals (e.g., amantadine, rimantadine, gancyclovir,acyclovir, ribavirin, penciclovir, oseltamivir, foscarnet zidovudine(AZT), didanosine (ddI), lamivudine (3TC), zalcitabine (ddC), stavudine(d4T), nevirapine, delavirdine, indinavir, ritonavir, vidarabine,nelfinavir, saquinavir, relenza, tamiflu, pleconaril, interferons,etc.), steroids and corticosteroids such as prednisone, cortisone,fluticasone and glucocorticoid, antibiotics, analgesics,bronchodialaters, or other treatments for respiratory and/or viralinfections), thereby inhibiting infection and replication of the hSARSvirus. In addition, the present invention provides methods for treating,ameliorating, managing, or preventing SARS by administering the vaccinepreparations or antibodies of the present invention alone or incombination with antivirals.

Furthermore, the present invention provides pharmaceutical compositionscomprising anti-viral agents of the present invention and apharmaceutically acceptable carrier. The present invention also provideskits comprising pharmaceutical compositions of the present invention.

5.2 RNA Interference

The present invention specifically relates to the use of gene therapy totreat SARS in humans. In certain embodiments, gene therapy is conductedwith the use of polynucleotide compounds, such as but not limited toantisense polynucleotides, ribozymes, RNA interference molecules, triplehelix polynucleotides and the like, where the nucleotide sequence ofsuch compounds are related to the nucleotide sequences of DNA and/or RNAof genes that are linked to the initiation mRNA transciption.

Antisense technology has been the most commonly described approach inprotocols to achieve gene-specific interference. For antisensestrategies, stoichiometric amounts of single-stranded nucleic acidcomplementary to the messenger RNA for the gene of interest areintroduced into the cell. See U.S. Pat. No. 6,506,559, which isincorporated herein by reference in its entirety. Triple helical nucleicacid structures are also useful for engineered interference. Thisapproach relies on the rare ability of certain nucleic acid populationsto adopt a triple-stranded structure. Under physiological conditions,nucleic acids are virtually all single- or double-stranded, and rarelyif ever form triple-stranded structures. It has been known for sometime, however, that certain simple purine- or pyrimidine-rich sequencescould form a triple-stranded molecule in vitro under extreme conditionsof pH (i.e., in a test tube). Such structures are generally verytransient under physiological conditions, so that simple delivery ofunmodified nucleic acids designed to produce triple-strand structuresdoes not yield interference.

In certain embodiments, an RNA interference (RNAi) molecule is used todecrease or inhibit expression of the nucleic acid against which theRNAi is directed. RNAi refers to the use of double-stranded RNA (dsRNA)or small interfering RNA (siRNA) to suppress the expression of a genecomprising a related nucleotide sequence. RNAi is also calledpost-transcriptional gene silencing (or PTGS). Since the only RNAmolecules normally found in the cytoplasm of a cell are molecules ofsingle-stranded mRNA, the cell has enzymes that recognize and cut dsRNAinto fragments containing 21-25 base pairs (approximately two turns of adouble helix and which are referred to as small interfering RNA orsiRNA). The antisense strand of the fragment separates enough from thesense strand so that it hybridizes with the complementary sense sequenceon a molecule of endogenous cellular mRNA. This hybridization triggerscutting of the mRNA in the double-stranded region, thus destroying itsability to be translated into a polypeptide. Introducing dsRNAcorresponding to a particular gene thus knocks out the cell's ownexpression of that gene in particular tissues and/or at a chosen time.Thus, RNAi regulates gene expression via a ubiquitous mechanism bydegradation of target mRNA in a sequence-specific manner. McManus etal., 2002, Nat Rev Genet 3:737-747. In mammalian cells, interfering RNA(RNAi) can be triggered by 21- to 23-nucleotide duplexes of siRNA. Leeet al., 2002, Nat Biotechnol 20: 500-505; Paul et al., 2002, NatBiotechnol. 20:505-508; Miyagishi et al., 2002, Nat Biotechnol.20:497-500; Paddison et al., 2002, Genes Dev. 16: 948-958. Theexpression of siRNA or short hairpin RNA (shRNA) driven by U6 promotereffectively mediates target mRNA degradation in mammalian cells.Synthetic siRNA duplexes and plasmid-derived siRNAs can inhibit HIV-1infection and replication by specifically degrading HIV genomic RNA.McManus et al., J. Immunol. 169:5754-5760; Jacque et al., 2002, Nature418:435-438; Novina et al., 2002, Nat Med 8:681-686. Also, siRNAtargeting HCV genomic RNA inhibits HCV replication. Randall et al.,2003, Proc Natl Acad Sci USA 100:235-240; Wilson et al., 2003, Proc NatlAcad Sci USA 100: 2783-2788. Fas targeted by siRNA protects the liverfrom fulminant hepatitis and fibrosis. Song et al., 2003, Nat Med9:347-351. However, the possibility that RNA interference might inhibithSARS viral replication has not been known until the present invention.

Double-stranded (ds) RNA can be used to interfere with gene expressionin many organisms including, but not limited to mammals. dsRNA is usedas inhibitory RNA or RNAi of the function of a nucleic acid molecule ofthe invention to produce a phenotype that is the same as that of a nullmutant of a nucleic acid molecule of the invention (Wianny &Zernicka-Goetz, 2000, Nature Cell Biology 2: 70-75).

Many methods have been developed to make siRNA, e.g., chemical synthesisor in vitro transcription. Once made, the siRNA can be introduceddirectly into a cell to mediate RNA interference (Elbashir et al., 2001,Nature 411: 494-498; Song, E, et al. RNA interference targeting Fasprotects mice from fulminant hepatitis. Nat. Med. 2003; 9: 347-351; andLewis, D L, et al. Efficient delivery of siRNA for inhibition of geneexpression in postnatal mice. Nat. Genet. 2002; 32: 107-108).Alternatively, the siRNA can be encapsulated into liposomes tofacilitate delivery into a cell (Sorensen, D R, et al. Gene silencing bysystemic delivery of synthetic siRNAs in adult mice. J Mol Biol. 2003;327: 761-766). The siRNAs can also be introduced into cells viatransient transfection. See also U.S. patent application Nos. 60/265232and 09/821832, and International Application No. PCT/US01/10188,directed to RNA sequence-specific mediators of RNA interference.

A number of expression vectors have also been developed to continuallyexpress siRNAs in transiently and stably transfected mammalian cells(Brummelkamp et al., 2002, Science 296: 550-553; Sui et al., 2002, PNAS99(6): 5515-5520; Paul et al., 2002, Nature Biotechnol. 20: 505-508).Some of these vectors have been engineered to express small hairpin RNAs(shRNAs), which get processed in vivo into siRNA-like molecules capableof carrying out gene-specific silencing. In certain embodiments, anshRNA contains plasmid under the control of a promoter, preferably a U6promoter (Paul, CP, et al. Effective expression of small interfering RNAin human cells. Nat. Biotechnol. 2002; 20: 505-508). Another type ofsiRNA expression vector encodes the sense and antisense siRNA strandsunder control of separate pol III promoters (Miyagishi and Taira, 2002,Nature Biotechnol. 20: 497-500). The siRNA strands from this vector,like the shRNAs of the other vectors, have 3′ thymidine terminationsignals. The shRNA gene can be delivered via a suitable vector system,e.g., adenovirus, adeno-associated virus (AAV), or retrovirus (Xia, H,et al. siRNA-mediated gene silencing in vitro and in vivo. Nat.Biotechnol. 2002; 20: 1006-1010; and Barton, G M, et al. Retroviraldelivery of small interfering RNA into primary cells. Proc. Natl. Acad.Sci. USA 2002; 99: 14943-14945). Silencing efficacy by both types ofexpression vectors is comparable to that induced by transientlytransfecting siRNA.

The RNA may comprise one or more strands of polymerized ribonucleotide.It may include modifications to either the phosphate-sugar backbone orthe nucleoside. For example, the phophodiester linkages of natural RNAmay be modified to include at least one of a nitrogen or sulfurheteroatom. Modifications in RNA structure may be tailored to allowspecific genetic inhibition while avoiding a general panic response insome organisms which is generated by dsRNA. Likewise, bases may bemodified to block the activity of adenosine deaminase. RNA may beproduced enzymatically or by partial/total organic synthesis; anymodified ribonucleotide can be introduced by in vitro enzymatic ororganic synthesis.

The double-stranded structure may be formed by a singleself-complementary RNA strand or two complementary RNA strands. RNAduplex formation may be initiated either inside or outside the cell. TheRNA may be introduced in an amount which allows delivery of at least onecopy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of double-stranded material may yield more effectiveinhibition; lower doses may also be useful for specific applications.Inhibition is sequence-specific in that nucleotide sequencescorresponding to the duplex region of the RNA are targeted for geneticinhibition. The RNA molecule may be at least 10, 12, 15, 20, 21, 22, 23,24, 25 or 30 nucleotides in length.

siRNAs of the present invention specifically target the region of SCoVviral RNA species encoding Replicase 1A, S glycoprotein, E protein, Mprotein and N protein, respectively (FIGS. 1A and 4A). Thenon-structural rep gene products play key roles in viral replication andgene transcription. The structure proteins play critical roles in viralentry (S), package (E, N), and secretion (M). It is particularlyimportant to develop effective siRNAs targeting structure genes, becausedrugs used in combination with multiple targets usually exhibit enhancedantiviral effects and eliminate drug resistant mutations.

RNA containing a nucleotide sequences identical to a portion of thetarget gene are preferred for inhibition. RNA sequences with insertions,deletions, and single point mutations relative to the target sequencehave also been found to be effective for inhibition. Thus, sequenceidentity may optimized by sequence comparison and alignment algorithmsknown in the art (see Gribskov and Deveeux, Sequence Analysis primer,Stockton Press, 1991, and references cited therein) and calculating thepercent difference between the nucleotide sequences by, for example, thesmith-Waterman algorithm as implemented in the BESTFIT software programusing default parameters (e.g., University of Wisconsin GeneticComputing Group). Greater than 90% sequence identity, or even 100%sequence identity, between the inhibitory RNA and the portion of thetarget gene is preferred. Alternatively, the duplex region of the RNAmay be defined functionally as a nucleotide sequence that is capable ofhybridizing with a portion of the target gene transcript (e.g., 400 mMNaCl, 40 mM PIPES, pH6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for12-16 hours; followed by washing). The length of the identicalnucleotide sequences may be at least 10, 25, 50, 100, 200, 300 or 400bases.

One hundred percent sequence identity between the RNA and the targetgene is not required to practice the present invention. Thus, theinvention has the advantage of being able to tolerate sequencevariations that might be expected due to genetic mutation, strainpolymorphism, or evolutionary divergence.

RNA may be synthesized either in vivo or in vitro. Endogenous RNApolymerase of the cell may mediate transcription in vivo, or cloned RNApolymerase can be used for transcription in vivo or in vitro. Fortranscription from a transgene in vivo or an expression construct, aregulatory region (e.g., promoter, enhancer, silencer, splice donor andacceptor, polyadenylation) may be used to transcribe the RNA strand (orstrands). Inhibition may be targeted by specific transcription in anorgan, tissue, or cell type; stimulation of an environmental condition(e.g., infection, stress, temperature, chemical inducers); and/orengineering transcription at a developmental stage or age. The RNAstrands may or may not be polyadenylated; the RNA strands may or may notbe capable of being translated into a polypeptide by a cell'stranslational apparatus. RNA may be chemically or enzymaticallysynthesized by manual or automated reactions. The RNA may be synthesizedby a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g.,T3, T7, SP6). The use and production of an expression construct areknown in the art (see also WO 97/32016; U.S. Pat. Nos. 5,593,874,5,698,425, 5,712,135, 5,789,214, and 5,804,693; and the references citedtherein). If synthesized chemically or by in vitro enzymatic synthesis,the RNA may be purified prior to introduction into the cell. Forexample, RNA can be purified from a mixture by extraction with a solventor resin, precipitation, electrophoresis, chromatography, or acombination thereof. Alternatively, the RNA may be used with no or aminimum of purification to avoid losses due to sample processing. TheRNA may be dried for storage or dissolved in an aqueous solution. Thesolution may contain buffers or salts to promote annealing, and/orstabilization of the duplex strands.

RNA may be directly introduced into the cell (i.e., intracellularly); orintroduced extracellularly into a cavity, interstitial space, into thecirculation of an organism, introduced orally, or may be introduced bybathing an organism in a solution containing the RNA. Physical methodsof introducing nucleic acids, for example, injection directly into thecell or extracellular injection into the organism, may also be used.Vascular or extravascular circulation, the blood or lymph system, andthe cerebrospinal fluid are sites where the RNA may be introduced.

Physical methods of introducing nucleic acids include injection of asolution containing the RNA, bombardment by particles covered by theRNA, soaking the cell or organism in a solution of the RNA, orelectroporation of cell membranes in the presence of the RNA. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. Other methods known in theart for introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, such ascalcium phosphate, and the like. Thus the RNA may be introduced alongwith components that perform one or more of the following activities:enhance RNA uptake by the cell, promote annealing of the duplex strands,stabilize the annealed strands, or other-wise increase inhibition of thetarget gene.

RNAi technology has been adapted for high throughput use in C. elegans(see, e.g., Kamath et al., 2003, Nature 421: 231-7, Ashrafi et al.,2003, Nature 421: 268-72, Taschl, 2003, Nature 421: 220-221). Briefly,DNA plasmids encoding a double-stranded RNA (dsRNA) of choice areinserted into E. coli. The nucleic acid encoding the dsRNA can be placedunder the control of an inducible promoter such that expression in E.coli occurs only in the presence of the inducing molecule (e.g., IPTG).Nematodes at the latest larval stage are placed on a lawn of E. coliexpressing the dsRNA and allowed to feed on the E. coli. The ingestedbacteria release the dsRNA inside the nematode. As a result, the genewhose sequence corresponds to that of the dsRNA behaves as if the genecarries a loss-of-function mutation.

In the present invention, siRNAs may be combined with other anti-viralagents to increase its anti-SCoV efficacy. Furthermore, the siRNAs ofthe present invention that target different sites of the gene regions ordifferent RNA species of SCoV, may be combined with one another.

Accordingly, the present invention provides methods of inhibiting hSARSinfection, or replication in a cell by administering to the cell aneffective amount of the nucleic acid molecule comprising or,alternatively consisting of the nucleotide sequence of SEQ ID NO:1, 2,3, 4, 5, 6, 7, 8, 9, 10 and/or 11, or a complement thereof, or a portionthereof. Furthermore, the present invention provides methods ofpreventing, ameliorating, treating or managing SARS by administering toa subject in need thereof a therapeutically or prophylacticallyeffective amount of the nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and/or 11, or acomplement thereof, or a portion thereof.

5.3 Recombinant and Chimeric hSARS Viruses

The present invention encompasses recombinant or chimeric virusesencoded by viral vectors derived from the genome of hSARS virus ornatural variants thereof. In a specific embodiment, the virus has agenome comprising the nucleotide sequence of SEQ ID NO:1, 2, 3, 4, 5, 6,7, 8, 9, 10 and/or 11, or a portion thereof, or a variant thereof, dueto one or more naturally occurred mutations or by recombinant DNAtechnology, including, but not limited to, point mutations,rearrangements, insertions, deletions etc., to the genomic sequence thatmay or may not result in a phenotypic change. In accordance with thepresent invention, a viral vector which is derived from the genome ofthe hSARS virus, is one that contains a nucleic acid sequence thatencodes at least a part of one ORF of the hSARS virus. In a specificembodiment, the ORF comprises or consists of a nucleotide sequence ofSEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, or a fragment thereof. Ina specific embodiment, there are more than one ORF within the nucleotidesequence of the hSARS genome, or a fragment thereof. In accordance withthe present invention these viral vectors may or may not include nucleicacids that are non-native to the viral genome.

In another specific embodiment, a chimeric virus of the invention is arecombinant hSARS virus which further comprises a heterologousnucleotide sequence. In accordance with the invention, a chimeric virusmay be encoded by a nucleotide sequence in which heterologous nucleotidesequences have been added to the genome or in which endogenous or nativenucleotide sequences have been replaced with heterologous nucleotidesequences.

According to the present invention, the chimeric viruses are encoded bythe viral vectors of the invention which further comprise a heterologousnucleotide sequence. In accordance with the present invention a chimericvirus is encoded by a viral vector that may or may not include nucleicacids that are non-native to the viral genome. In accordance with theinvention a chimeric virus is encoded by a viral vector to whichheterologous nucleotide sequences have been added, inserted orsubstituted for native or non-native sequences. In accordance with thepresent invention, the chimeric virus may be encoded by nucleotidesequences derived from different strains or variants of hSARS virus. Inparticular, the chimeric virus is encoded by nucleotide sequences thatencode antigenic polypeptides derived from different strains or variantsof hSARS virus.

A chimeric virus may be of particular use for the generation ofrecombinant vaccines protecting against two or more viruses (Tao et al.,J. Virol. 72: 2955-2961; Durbin et al., 2000, J. Virol. 74: 6821-6831;Skiadopoulos et al., 1998, J. Virol. 72: 1762-1768; Teng et al., 2000,J. Virol. 74: 9317-9321). For example, it can be envisaged that a virusvector derived from the hSARS virus expressing one or more proteins ofvariants of hSARS virus, or vice versa, will protect a subjectvaccinated with such vector against infections by both the native hSARSand the variant. Attenuated and replication-defective viruses may be ofuse for vaccination purposes with live vaccines as has been suggestedfor other viruses. (See, PCT WO 02/057302, at pp. 6 and 23, incorporatedby reference herein).

In accordance with the present invention the heterologous sequence to beincorporated into the viral vectors encoding the recombinant or chimericviruses of the invention include sequences obtained or derived fromdifferent strains or variants of hSARS.

In certain embodiments, the chimeric or recombinant viruses of theinvention are encoded by viral vectors derived from viral genomeswherein one or more sequences, intergenic regions, termini sequences, orportions or entire ORF have been substituted with a heterologous ornon-native sequence. In certain embodiments of the invention, thechimeric viruses of the invention are encoded by viral vectors derivedfrom viral genomes wherein one or more heterologous sequences have beeninserted or added to the vector.

The selection of the viral vector may depend on the species of thesubject that is to be treated or protected from a viral infection. Ifthe subject is human, then an attenuated hSARS virus can be used toprovide the antigenic sequences.

In accordance with the present invention, the viral vectors can beengineered to provide antigenic sequences which confer protectionagainst infection by the hSARS and natural variants thereof. The viralvectors may be engineered to provide one, two, three or more antigenicsequences. In accordance with the present invention the antigenicsequences may be derived from the same virus, from different strains orvariants of the same type of virus, or from different viruses.

The expression products and/or recombinant or chimeric virions obtainedin accordance with the invention may advantageously be utilized invaccine formulations. The expression products and chimeric virions ofthe present invention may be engineered to create vaccines against abroad range of pathogens, including viral and bacterial antigens, tumorantigens, allergen antigens, and auto antigens involved in autoimmunedisorders. In particular, the chimeric virions of the present inventionmay be engineered to create vaccines for the protection of a subjectfrom infections with hSARS virus and variants thereof.

In certain embodiments, the expression products and recombinant orchimeric virions of the present invention may be engineered to createvaccines against a broad range of pathogens, including viral antigens,tumor antigens and autoantigens involved in autoimmune disorders. Oneway to achieve this goal involves modifying existing hSARS genes tocontain foreign sequences in their respective external domains. Wherethe heterologous sequences are epitopes or antigens of pathogens, thesechimeric viruses may be used to induce a protective immune responseagainst the disease agent from which these determinants are derived.

Thus, the present invention relates to the use of viral vectors andrecombinant or chimeric viruses to formulate vaccines against a broadrange of viruses and/or antigens. The present invention also encompassesrecombinant viruses comprising a viral vector derived from the hSARS orvariants thereof which contains sequences which result in a virus havinga phenotype more suitable for use in vaccine formulations, e.g.,attenuated phenotype or enhanced antigenicity. The mutations andmodifications can be in coding regions, in intergenic regions and in theleader and trailer sequences of the virus.

The invention provides a host cell comprising a nucleic acid or a vectoraccording to the invention. Plasmid or viral vectors containing thepolymerase components of hSARS virus are generated in prokaryotic cellsfor the expression of the components in relevant cell types (bacteria,insect cells, eukaryotic cells). Plasmid or viral vectors containingfull-length or partial copies of the hSARS genome will be generated inprokaryotic cells for the expression of viral nucleic acids in-vitro orin-vivo. The latter vectors may contain other viral sequences for thegeneration of chimeric viruses or chimeric virus proteins, may lackparts of the viral genome for the generation of replication defectivevirus, and may contain mutations, deletions or insertions for thegeneration of attenuated viruses.

Infectious copies of hSARS (being wild type, attenuated,replication-defective or chimeric) can be produced upon co-expression ofthe polymerase components according to the state-of-the-art technologiesdescribed above.

In addition, the present invention provides eukaryotic cells,transiently or stably expressing one or more full-length or partialhSARS viral nucleic acids or proteins. Such cells can be made bytransfection (proteins or nucleic acid vectors), infection (viralvectors) or transduction (viral vectors) and may be useful forcomplementation of mentioned wild type, attenuated,replication-defective or chimeric viruses.

Accordingly, the present invention further provides a host cell that istransfected or transduced with the nucleic acid molecules of the presentinvention or infected with the chimeric viruses of the presentinvention.

The viral vectors and chimeric viruses of the present invention may beused to modulate a subject's immune system by stimulating a humoralimmune response, a cellular immune response or by stimulating toleranceto an antigen. As used herein, a subject means: humans, primates,horses, cows, sheep, pigs, goats, dogs, cats, avian species and rodents.

5.4 Formulation of Vaccines

In a preferred embodiment, the invention provides a proteinaceousmolecule or hSARS virus specific viral protein or functional fragmentthereof encoded by a nucleic acid according to the invention. Usefulproteinaceous molecules are for example derived from any of the genes orgenomic fragments derivable from the virus according to the invention,including envelop protein (E protein), integral membrane protein (Mprotein), spike protein (S protein), nucleocapsid protein (N protein),hemaglutinin esterase (HE protein), and RNA-dependent RNA polymerase.Such molecules, or antigenic fragments thereof, as provided herein, arefor example useful in diagnostic methods or kits and in pharmaceuticalcompositions such as subunit vaccines. Particularly useful arepolypeptides encoded by the nucleotide sequence of SEQ ID NO:1, 2, 3, 4,5, 6, 7, 8, 9, 10 or 11, or antigenic fragments thereof for inclusion asantigen or subunit immunogen. Particularly useful are also thoseproteinaceous substances that are encoded by recombinant nucleic acidfragments of the hSARS genome, of course preferred are those that arewithin the preferred bounds and metes of ORFs, in particular, foreliciting hSARS specific antibody or T cell responses, whether in vivo(e.g., for protective or therapeutic purposes or for providingdiagnostic antibodies) or in vitro (e.g., by phage display technology oranother technique useful for generating synthetic antibodies).

The invention provides vaccine formulations for the prevention andtreatment of infections with hSARS virus. In certain embodiments, thevaccine of the invention comprises recombinant and chimeric viruses ofthe hSARS virus, comprising a nucleotide sequence of SEQ ID NO:1, 2, 3,4, 5, 6, 7, 8, 9, 10 and/or 11.

The vaccine of the present invention may be formulated with a suitableadjuvant in order to enhance the immunological response. Such adjuvantsmay include but are not limited to mineral gels, e.g., aluminumhydroxide; surface active substances such as lysolecithin, pluronicpolyols, polyanions; peptides; oil emulsions; and potentially usefulhuman adjuvants such as BCG and Corynebacterium parvum.

In another aspect, the present invention also provides DNA vaccineformulations comprising nucleic acid molecules having the sequence ofSEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and/or 11, or a fragmentthereof. In another specific embodiment, the DNA vaccine formulations ofthe present invention comprises a nucleic acid or fragment thereofencoding the antibodies which immunospecifically binds hSARS viruses. InDNA vaccine formulations, a vaccine DNA comprises a viral vector, suchas that derived from the hSARS virus, bacterial plasmid, or otherexpression vector, bearing an insert comprising a nucleic acid moleculeof the present invention operably linked to one or more controlelements, thereby allowing expression of the vaccinating proteinsencoded by said nucleic acid molecule in a vaccinated subject. Suchvectors can be prepared by recombinant DNA technology as recombinant orchimeric viral vectors carrying a nucleic acid molecule of the presentinvention (see also Section 5, supra).

Various heterologous vectors are described for DNA vaccinations againstviral infections. For example, the vectors described in the followingreferences may be used to express hSARS sequences instead of thesequences of the viruses or other pathogens described; in particular,vectors described for hepatitis B virus (Michel, M. L. et al., 1995,DAN-mediated immunization to the hepatitis B surface antigen in mice:Aspects of the humoral response mimic hepatitis B viral infection inhumans, Proc. Natl. Acta. Sci. USA 92: 5307-5311; Davis, H. L. et al.,1993, DNA-based immunization induces continuous seretion of hepatitis Bsurface antigen and high levels of circulating antibody, Human Molec.Genetics 2: 1847-1851), HIV virus (Wang, B. et al., 1993, Geneinoculation generates immune responses against human immunodeficiencyvirus type 1, Proc. Natl. Acad. Sci. USA 90: 4156-4160; Lu, S. et al.,1996, Simian immunodeficiency virus DNA vaccine trial in macques, J.Virol. 70: 3978-3991; Letvin, N. L. et al., 1997, Potent, protectiveanti-HIV immune responses generated by bimodal HIV envelope DNA plusprotein vaccination, Proc Natl Acad Sci USA. 94(17): 9378-83), andinfluenza viruses (Robinson, H L et al., 1993, Protection against alethal influenza virus challenge by immunization with ahaemagglutinin-expressing plasmid DNA, Vaccine 11: 957-960; Ulmer, J. B.et al., Heterologous protection against influenza by injection of DNAencoding a viral protein, Science 259: 1745-1749), as well as bacterialinfections, such as tuberculosis (Tascon, R. E. et al., 1996,Vaccination against tuberculosis by DNA injection, Nature Med. 2:888-892; Huygen, K. et al., 1996, Immunogenicity and protective efficacyof a tuberculosis DNA vaccine, Nature Med, 2: 893-898), and parasiticinfection, such as malaria (Sedegah, M., 1994, Protection againstmalaria by immunization with plasmid DNA encoding circumsporozoiteprotein, Proc. Natl. Acad. Sci. USA 91: 9866-9870; Doolan, D. L. et al.,1996, Circumventing genetic restriction of protection against malariawith multigene DNA immunization: CD8+T cell-interferon δ, and nitricoxide-dependent immunity, J. Exper. Med., 1183: 1739-1746).

Many methods may be used to introduce the vaccine formulations describedabove. These include, but are not limited to, oral, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, andintranasal routes. Alternatively, it may be preferable to introduce thechimeric virus vaccine formulation via the natural route of infection ofthe pathogen for which the vaccine is designed. The DNA vaccines of thepresent invention may be administered in saline solutions by injectionsinto muscle or skin using a syringe and needle (Wolff J. A. et al.,1990, Direct gene transfer into mouse muscle in vivo, Science 247:1465-1468; Raz, E., 1994, Intradermal gene immunization: The possiblerole of DNA uptake in the induction of cellular immunity to viruses,Proc. Natl. Acd. Sci. USA 91: 9519-9523). Another way to administer DNAvaccines is called “gene gun” method, whereby microscopic gold beadscoated with the DNA molecules of interest is fired into the cells (Tang,D. et al., 1992, Genetic immunization is a simple method for elicitingan immune response, Nature 356: 152-154). For general reviews of themethods for DNA vaccines, see Robinson, H. L., 1999, DNA vaccines: basicmechanism and immune responses (Review), Int. J. Mol. Med. 4(5):549-555; Barber, B., 1997, Introduction: Emerging vaccine strategies,Seminars in Immunology 9(5): 269-270; and Robinson, H. L. et al., 1997,DNA vaccines, Seminars in Immunology 9(5): 271-283.

5.5 Adjuvants and Carrier Molecules

hSARS-associated antigens are administered with one or more adjuvants.In one embodiment, the hSARS-associated antigen is administered togetherwith a mineral salt adjuvants or mineral salt gel adjuvant. Such mineralsalt and mineral salt gel adjuvants include, but are not limited to,aluminum hydroxide (ALHYDROGEL, REHYDRAGEL), aluminum phosphate gel,aluminum hydroxyphosphate (ADJU-PHOS), and calcium phosphate.

In another embodiment, hSARS-associated antigen is administered with animmunostimulatory adjuvant. Such class of adjuvants, include, but arenot limited to, cytokines (e.g., interleukin-2, interleukin-7,interleukin-12, granulocyte-macrophage colony stimulating factor(GM-CSF), interfereon-γ interleukin-1β (IL-1β), and IL-1β peptide orSclavo Peptide), cytokine-containing liposomes, triterpenoid glycosidesor saponins (e.g., QuilA and QS-21, also sold under the trademarkSTIMULON, ISCOPREP), Muramyl Dipeptide (MDP) derivatives, such asN-acetyl-muramyl-L-threonyl-D-isoglutamine (Threonyl-MDP, sold under thetrademark TERMURTIDE), GMDP,N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine,N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine, muramyl tripeptide phosphatidylethanolamine(MTP-PE), unmethylated CpG dinucleotides and oligonucleotides, such asbacterial DNA and fragments thereof, LPS, monophosphoryl Lipid A (3D-MLAsold under the trademark MPL), and polyphosphazenes.

In another embodiment, the adjuvant used is a particular adjuvant,including, but not limited to, emulsions, e.g., Freund's CompleteAdjuvant, Freund's Incomplete Adjuvant, squalene or squalaneoil-in-water adjuvant formulations, such as SAF and MF59, e.g., preparedwith block-copolymers, such as L-121 (polyoxypropylene/polyoxyetheylene)sold under the trademark PLURONIC L-121, Liposomes, Virosomes,cochleates, and immune stimulating complex, which is sold under thetrademark ISCOM.

In another embodment, a microparticular adjuvant is used.,Microparticulare adjuvants include, but are not limited to biodegradableand biocompatible polyesters, homo- and copolymers of lactic acid (PLA)and glycolic acid (PGA), poly(lactide-co-glycolides) (PLGA)microparticles, polymers that self-associate into particulates(poloxamer particles), soluble polymers (polyphosphazenes), andvirus-like particles (VLPs) such as recombinant protein particulates,e.g., hepatitis B surface antigen (HbsAg).

Yet another class of adjuvants that may be used include mucosaladjuvants, including but not limited to heat-labile enterotoxin fromEscherichia coli (LT), cholera holotoxin (CT) and cholera Toxin BSubunit (CTB) from Vibrio cholerae, mutant toxins (e.g., LTK63 andLTR72), microparticles, and polymerized liposomes.

In other embodiments, any of the above classes of adjuvants may be usedin combination with each other or with other adjuvants. For example,non-limiting examples of combination adjuvant preparations that can beused to administer the hSARS-associated antigens of the inventioninclude liposomes containing immunostimulatory protein, cytokines, orT-cell and/or B-cell peptides, or microbes with or without entrappedIL-2 or microparticles containing enterotoxin. Other adjuvants known inthe art are also included within the scope of the invention (see VaccineDesign: The Subunit and Adjuvant Approach, Chap. 7, Michael F. Powelland Mark J. Newman (eds.), Plenum Press, New York, 1995, which isincorporated herein in its entirety).

The effectiveness of an adjuvant may be determined by measuring theinduction of antibodies directed against an immunogenic polypeptidecontaining a hSARS polypeptide epitope, the antibodies resulting fromadministration of this polypeptide in vaccines which are also comprisedof the various adjuvants.

The polypeptides may be formulated into the vaccine as neutral or saltforms. Pharmaceutically acceptable salts include the acid additionalsalts (formed with free amino groups of the peptide) and which areformed with inorganic acids, such as, for example, hydrochloric orphosphoric acids, or organic acids such as acetic, oxalic, tartaric,maleic, and the like. Salts formed with free carboxyl groups may also bederived from inorganic bases, such as, for example, sodium potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,procaine and the like.

The vaccines of the invention may be multivalent or univalent.Multivalent vaccines are made from recombinant viruses that direct theexpression of more than one antigen.

Many methods may be used to introduce the vaccine formulations of theinvention; these include but are not limited to oral, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasalroutes, and via scarification (scratching through the top layers ofskin, e.g., using a bifurcated needle).

The patient to which the vaccine is administered is preferably a mammal,most preferably a human, but can also be a non-human animal includingbut not limited to cows, horses, sheep, pigs, fowl (e.g., chickens),goats, cats, dogs, hamsters, mice and rats.

5.6 Preparation of Antibodies

Antibodies which specifically recognize a polypeptide of the invention,such as, but not limited to, polypeptides encoded by the nucleotidesequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, or hSARSepitope, or antigen-binding fragments thereof can be used for detecting,screening, and isolating the polypeptide of the invention or fragmentsthereof, or similar sequences that might encode similar proteins orpolypeptides from the other organisms. Such antibodies can be used forvarious in vitro detection assays, including enzyme-linked immunosorbentassays (ELISA), radioimmunoassays, Western blot, etc., for the detectionof a polypeptide of the invention or, preferably, hSARS, in samples, forexample, a biological material, including cells, cell culture media(e.g., bacterial cell culture media, mammalian cell culture media,insect cell culture media, yeast cell culture media, etc.), blood,plasma, serum, tissues, sputum, naseopharyngeal aspirates, etc.

Antibodies specific for a polypeptide of the invention or any epitope ofhSARS may be generated by any suitable method known in the art.Polyclonal antibodies to an antigen-of-interest, for example, thepolypeptide encoded by the nucleotide sequence of SEQ ID NO:1, 2, 3, 4,5, 6, 7, 8, 9, 10 or 11, or a complement thereof, can be produced byvarious procedures well known in the art. For example, an antigen can beadministered to various host animals including, but not limited to,rabbits, mice, rats, etc., to induce the production of antiseracontaining polyclonal antibodies specific for the antigen. Variousadjuvants may be used to increase the immunological response, dependingon the host species, and include but are not limited to, Freund's(complete and incomplete) adjuvant, mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful adjuvants for humanssuch as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum. Suchadjuvants are also well known in the art. (See Section 5.5, supra).

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas, pp. 563-681 (Elsevier, N. Y., 1981) (both of which areincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In anon-limiting example, mice can be immunized with an antigen of interestor a cell expressing such an antigen. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well known techniques to any suitablemyeloma cells. Hybridomas are selected and cloned by limiting dilution.The hybridoma clones are then assayed by methods known in the art forcells that secrete antibodies capable of binding the antigen. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by inoculating mice intraperitoneally with positive hybridomaclones.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments may be producedby proteolytic cleavage of inmmunoglobulin molecules, using enzymes suchas papain (to produce Fab fragments) or pepsin (to produce F(ab′)₂fragments). F(ab′)₂ fragments contain the complete light chain, and thevariable region, the CH1 region and the hinge region of the heavy chain.

The antibodies of the invention or fragments thereof can be alsoproduced by any method known in the art for the synthesis of antibodies,in particular, by chemical synthesis or preferably, by recombinantexpression techniques.

The nucleotide sequence encoding an antibody may be obtained from anyinformation available to those skilled in the art (i.e., from Genbank,the literature, or by routine cloning and sequence analysis). If a clonecontaining a nucleic acid encoding a particular antibody or anepitope-binding fragment thereof is not available, but the sequence ofthe antibody molecule or epitope-binding fragment thereof is known, anucleic acid encoding the immunoglobulin may be chemically synthesizedor obtained from a suitable source (e.g., an antibody cDNA library, or acDNA library generated from, or nucleic acid, preferably poly A+RNA,isolated from any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody) by PCR amplificationusing synthetic primers hybridizable to the 3′ and 5′ ends of thesequence or by cloning using an oligonucleotide probe specific for theparticular gene sequence to identify, e.g., a cDNA clone from a cDNAlibrary that encodes the antibody. Amplified nucleic acids generated byPCR may then be cloned into replicable cloning vectors using any methodwell known in the art.

Once the nucleotide sequence of the antibody is determined, thenucleotide sequence of the antibody may be manipulated using methodswell known in the art for the manipulation of nucleotide sequences,e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.(see, for example, the techniques described in Sambrook et al., supra;and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,John Wiley & Sons, NY, which are both incorporated by reference hereinin their entireties), to generate antibodies having a different aminoacid sequence by, for example, introducing amino acid substitutions,deletions, and/or insertions into the epitope-binding domain regions ofthe antibodies or any portion of antibodies which may enhance or reducebiological activities of the antibodies.

Recombinant expression of an antibody requires construction of anexpression vector containing a nucleotide sequence that encodes theantibody. Once a nucleotide sequence encoding an antibody molecule or aheavy or light chain of an antibody, or portion thereof has beenobtained, the vector for the production of the antibody molecule may beproduced by recombinant DNA technology using techniques well known inthe art as discussed in the previous sections. Methods which are wellknown to those skilled in the art can be used to construct expressionvectors containing antibody coding sequences and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. The nucleotide sequenceencoding the heavy-chain variable region, light-chain variable region,both the heavy-chain and light-chain variable regions, anepitope-binding fragment of the heavy- and/or light-chain variableregion, or one or more complementarity determining regions (CDRs) of anantibody may be cloned into such a vector for expression. Thus-preparedexpression vector can be then introduced into appropriate host cells forthe expression of the antibody. Accordingly, the invention includes hostcells containing a polynucleotide encoding an antibody specific for thepolypeptides of the invention or fragments thereof.

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides or different selectablemarkers to ensure maintenance of both plasmids. Alternatively, a singlevector may be used which encodes, and is capable of expressing, bothheavy and light chain polypeptides. In such situations, the light chainshould be placed before the heavy chain to avoid an excess of toxic freeheavy chain (Proudfoot, 1986 Nature, 322: 52; and Kohler, 1980 Proc.Natl. Acad. Sci. USA 77: 2197-9). The coding sequences for the heavy andlight chains may comprise cDNA or genomic DNA.

In another embodiment, antibodies can also be generated using variousphage display methods known in the art. In phage display methods,functional antibody domains are displayed on the surface of phageparticles which carry the polynucleotide sequences encoding them. In aparticular embodiment, such phage can be utilized to display antigenbinding domains, such as Fab and Fv or disulfide-bond stabilized Fv,expressed from a repertoire or combinatorial antibody library (e.g.,human or murine). Phage expressing an antigen binding domain that bindsthe antigen of interest can be selected or identified with antigen,e.g., using labeled antigen or antigen bound or captured to a solidsurface or bead. Phage used in these methods are typically filamentousphage, including fd and M13. The antigen binding domains are expressedas a recombinantly fused protein to either the phage gene III or geneVIII protein. Examples of phage display methods that can be used to makethe immunoglobulins, or fragments thereof, of the present inventioninclude those disclosed in Brinkman et al., 1995 J. Immunol. Methods,182: 41-50; Ames et al., 1995 J. Immunol. Methods, 184: 177-186;Kettleborough et al., 1994 Eur. J. Immunol. 24: 952-958; Persic et al.,1997 Gene 187: 9-18; Burton et al., 1994 Advances in Immunology 57:191-280; PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired fragments, and expressed in any desired host, includingmammalian cells, insect cells, plant cells, yeast, and bacteria, e.g.,as described in detail below. For example, techniques to recombinantlyproduce Fab, Fab′ and F(ab′)2 fragments can also be employed usingmethods known in the art such as those disclosed in PCT publication WO92/22324; Mullinax et al., 1992, BioTechniques, 12(6): 864-869; andSawai et al., 1995 AJRI, 34: 26-34; and Better et al., 1988, Science240: 1041-1043 (each of which is incorporated by reference in itsentirety). Examples of techniques which can be used to producesingle-chain Fvs and antibodies include those described in U.S. Pat.Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology,203:46-88, 1991; Shu et al., PNAS, 90:7995-7999, 1993; and Skerra etal., 1988 Science 240: 1038-1040.

Once an antibody molecule of the invention has been produced by anymethods described above, it may then be purified by any method known inthe art for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A or Protein G purification, andsizing column chromatography), centrifugation, differential solubility,or by any other standard techniques for the purification of proteins.Further, the antibodies of the present invention or fragments thereofmay be fused to heterologous polypeptide sequences described herein orotherwise known in the art to facilitate purification.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use chimeric, humanized,or human antibodies. A chimeric antibody is a molecule in whichdifferent portions of the antibody are derived from different animalspecies, such as antibodies having a variable region derived from amurine monoclonal antibody and a constant region derived from a humanimmunoglobulin. Methods for producing chimeric antibodies are known inthe art. See e.g., Morrison, 1985 Science 229: 1202; Oi et al., 1986BioTechniques 4: 214; Gillies et al., 1989 J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which areincorporated herein by reference in their entireties. Humanizedantibodies are antibody molecules from non-human species that bind thedesired antigen having one or more complementarity determining regions(CDRs) from the non-human species and framework regions from a humanimmunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., 1988 Nature, 332: 323, which areincorporated herein by reference in their entireties. Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, 1991 Molecular Immunology, 28(4/5):489498; Studnicka et al., 1994 Protein Engineering 7(6): 805-814;Roguska et al., 1994 Proc Natl. Acad. Sci. USA, 91: 969-973), and chainshuffling (U.S. Pat. No. 5,565,332), all of which are herebyincorporated by reference in their entireties.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO96/34096; WO 96/33735; and WO 91/10741, each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For an overview of thistechnology for producing human antibodies, see Lonberg and Huszar, 1995Int. Rev. Immunol., 13: 65-93. For a detailed discussion of thistechnology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g., PCTpublications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735;European Pat. No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793;5,916,771; and 5,939,598, which are incorporated by reference herein intheir entireties. In addition, companies such as Abgenix, Inc. (Fremont,Calif.), Medarex (NJ) and Genpharm (San Jose, Calif.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (Jespers et al., 1988 Bio/technology, 12:899-903).

Antibodies fused or conjugated to heterologous polypeptides may be usedin vitro immunoassays and in purification methods (e.g., affinitychromatography) well known in the art. See e.g., PCT publication NumberWO 93/21232; EP 439,095; Naramura et al., 1994 Immunol. Lett. 39: 91-99;U.S. Pat. No. 5,474,981; Gillies et al., 1992 PNAS 89: 1428-1432; andFell et al., 1991 J. Immunol., 146: 2446-2452, which are incorporatedherein by reference in their entireties.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the polypeptidesof the invention or fragments, derivatives, analogs, or variantsthereof, or similar molecules having the similar enzymatic activities asthe polypeptide of the invention. Such solid supports include, but arenot limited to, glass, cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride or polypropylene.

5.7 Pharmaceutical Compositions and Kits

The present invention encompasses pharmaceutical compositions comprisinganti-viral agents of the present invention. In a specific embodiment,the anti-viral agent is an antibody which immunospecifically binds andneutralize the hSARS virus or variants thereof, or any proteins derivedtherefrom. In another specific embodiment, the anti-viral agent is apolypeptide or nucleic acid molecule of the invention. Thepharmaceutical compositions have utility as an anti-viral prophylacticagent and may be administered to a subject where the subject has beenexposed or is expected to be exposed to a virus.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu and Wu, 1987 J. Biol. Chem. 262: 4429 4432). Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds may be administered by any convenient route,for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Ina preferred embodiment, it may be desirable to introduce thepharmaceutical compositions of the invention into the lungs by anysuitable route. Pulmonary administration can also be employed, e.g., byuse of an inhaler or nebulizer, and formulation with an aerosolizingagent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. In oneembodiment, administration can be by direct injection at the site (orformer site) infected tissues.

In another embodiment, the pharmaceutical composition can be deliveredin a vesicle, in particular a liposome (see Langer, 1990 Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of InfectiousDisease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York,pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.).

In yet another embodiment, the pharmaceutical composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, 1987 CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980 Surgery 88: 507; and Saudek et al., 1989 N.Engl. J. Med. 321: 574). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, 1983 J. Macromol.Sci. Rev. Macromol. Chem. 23: 61; see also Levy et al., 1985 Science228: 190; During et al., 1989 Ann. Neurol. 25: 351; Howard et al., 1989,J. Neurosurg. 71: 105). In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, i.e., thelung, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, in Medical Applications of Controlled Release, supra, vol. 2,pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(1990 Science 249: 1527-1533).

The pharmaceutical compositions of the present invention comprise atherapeutically effective amount of a recombinant or chimeric hSARSvirus, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the pharmaceuticalcomposition is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained release formulations and the like. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. The formulation should suitthe mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The pharmaceutical compositions of the invention can be formulated asneutral or salt forms. Pharmaceutically acceptable salts include thoseformed with free amino groups such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withfree carboxyl groups such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2ethylamino ethanol, histidine, procaine, etc.

The amount of the pharmaceutical composition of the invention which willbe effective in the treatment of a particular disorder or condition willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. However, suitable dosage ranges forintravenous administration are generally about 20-500 micrograms ofactive compound per kilogram body weight. Suitable dosage ranges forintranasal administration are generally about 0.01 pg/kg body weight to1 mg/kg body weight. Effective doses may be extrapolated from doseresponse curves derived from in vitro or animal model test systems.

Suppositories generally contain active ingredient in the range of 0.5%to 10% by weight; oral formulations preferably contain 10% to 95% activeingredient.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. In apreferred embodiment, the kit contains an anti-viral agent of theinvention, e.g., an antibody immunospecific for the polypeptides encodedby a nucleotide sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or11, or any hSARS epitope, or a polypeptide or protein of the presentinvention, or a nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and/or 11, alone or incombination with adjuvants, antivirals, antibiotics, analgesic,bronchodialaters, or other pharmaceutically acceptable excipients.

5.8 Demonstration of Therapeutic Utility

The protocols and compositions of the invention are preferably tested invitro, and then in vivo, for the desired therapeutic or prophylacticactivity, prior to use in humans. For example, in vitro assays which canbe used to determine whether administration of a specific therapeuticprotocol is indicated, include in vitro cell culture assays in which apatient tissue sample is grown in culture, and exposed to or otherwiseadministered a protocol, and the effect of such protocol upon the tissuesample is observed. A lower level of cytopathic effects or survival ofthe contacted cells indicates that the therapeutic agent is effective totreat the condition in the patient. Alternatively, instead of culturingcells from a patient, therapeutic agents and methods may be screenedusing established cell lines, such as FRhK-4 (fetal rhesus monkeykidney) cells which are infected by hSARS virus (see Section 6, infra).Compounds for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to inrats, mice, chicken, cows, monkeys, rabbits, hamsters, etc.

The principle animal models for known in the art and widely used areknown and described in the art as described above.

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of thecombinatorial therapies disclosed herein for treatment or prevention ofSARS.

5.9 Detection Assays

The present invention provides a method for detecting an antibody, whichimmunospecifically binds to the hSARS virus or polypeptides of theinvention, in a biological sample, for example blood, serum, plasma,saliva, urine, etc., from a patient suffering from SARS. In a specificembodiment, the method comprising contacting the sample with thepolypeptides encoded by the nucleotide sequence of SEQ ID NO:1, 2, 3, 4,5, 6, 7, 8, 9, 10 and/or 11, directly immobilized on a substrate anddetecting the polypeptide-bound antibody directly or indirectly by alabeled heterologous anti-isotype antibody. In another specificembodiment, the sample is contacted with a host cell which istransfected or infected by the viral vector comprising the nucleotidesequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and/or 11 and thebound antibody can be detected by imrnunofluorescent assay.

The present invention provides a method for detecting the presence orabsence of a polypeptide or nucleic acid of the invention in abiological sample, comprising obtaining a biological sample from varioussources and contacting the sample with a compound or an agent capable ofdetecting an epitope or nucleic acid (e.g., mRNA, genomic DNA) of theinvention such that the presence of the hSARS virus is detected in thesample. A preferred agent for detecting hSARS mRNA or genomic RNA of theinvention is a labeled nucleic acid probe capable of hybridizing to mRNAor genomic RNA encoding a polypeptide of the invention or a complementthereof. The nucleic acid probe can be, for example, a nucleic acidmolecule comprising or consisting of the nucleotide sequence or SEQ IDNO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, or a portion thereof, or acomplement thereof, such as an oligonucleotide of at least 5, 10, 15 or20 or more contiguous nucleotides in length and sufficient tospecifically hybridize under stringent conditions to a hSARS mRNA orgenomic RNA.

In another preferred specific embodiment, the presence of hSARS virus isdetected in the sample by an reverse transcription polymerase chainreaction (RT-PCR) using the primers that are constructed based on apartial nucleotide sequence of the genome of hSARS virus, or anucleotide sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, aportion thereof, or a complement thereof. In a non-limiting specificembodiment, preferred primers to be used in a RT-PCR method are:5′-TACACACCTCAGCGTTG-3′ (SEQ ID NO:13) and 5′-CACGAACGTGACGAAT-3′ (SEQID NO:14)(Poon et al., 2003, Clin. Chem. 49(7):1-3, which isincorporated herein by reference in its entirety), in the presence of2.5 mM MgCl₂ and the thermal cycles are, for example, but not limitedto, 94° C. for 8 min followed by 40 cycles of 94° C. for 1 min, 50° C.for 1 min, 72° C. for 1 min. In more preferred specific embodiment, thepresent invention provides a real-time quantitative PCR assay to detectthe presence of hSARS virus in a biological sample by subjecting thecDNA obtained by reverse transcription of the extracted total RNA fromthe sample to PCR reactions using the specific primers, such as thosehaving nucleotide sequences of SEQ ID NOS:13 and 14, and a fluorescencedye, such as SYBR® Green I, which fluoresces when bound non-specificallyto double-stranded DNA. The fluorescence signals from these reactionsare captured at the end of extension steps as PCR product is generatedover a range of the thermal cycles, thereby allowing the quantitativedetermination of the viral load in the sample based on an amplificationplot. Alternatively, the real-time quantitative PCR may be performed bya TaqMan® assay using specific pairs of primers. The amplified productis detected by a TaqMan® probe which is labeled by a fluorescent dye anda quencher. The preferred primers to be used are: the forward primer;5′-GCTTAGGCCCTT TGAGAGAGACA -3′ SEQ ID NO:15); and the reverse primer,5′-GCCAATGCCAGTAG TGGTGTAAA-3′ (SEQ ID NO:16). In this case, theamplified product is detected by a probe, preferably having a nucleotidesequence 5′-(TET®)CTAATGTGCCTTTCTCCCCT GATG GCA(TAMRA®)-3′ (SEQ IDNO:17).

A preferred agent for detecting hSARS or the polypeptides of theinvention is an antibody that specifically binds a polypeptide of theinvention or any hSARS epitope, preferably an antibody with a detectablelabel. Antibodies can be polyclonal, or more preferably, monoclonal. Anintact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can beused.

The term “labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin. Thedetection method of the invention can be used to detect mRNA, protein(or any epitope), or genomic RNA in a sample in vitro as well as invivo. For example, in vitro techniques for detection of mRNA includenorthern hybridizations, in situ hybridizations, RT-PCR, and RNaseprotection. In vitro techniques for detection of an epitope of hSARS orthe polypeptides of the invention include enzyme linked immunosorbentassays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence. Furthermore, in vivo techniques for detection ofhSARS include introducing into a subject organism a labeled antibodydirected against the polypeptide. For example, the antibody can belabeled with a radioactive marker whose presence and location in thesubject organism can be detected by standard imaging techniques,including autoradiography.

In a specific embodiment, the methods further involve obtaining acontrol sample from a control subject, contacting the control samplewith a compound or agent capable of detecting hSARS, e.g., a polypeptideof the invention or mRNA or genomic RNA encoding a polypeptide of theinvention, such that the presence of hSARS or the polypeptide or mRNA orgenomic RNA encoding the polypeptide is detected in the sample, andcomparing the presence of hSARS or the polypeptide or mRNA or genomicRNA encoding the polypeptide in the control sample with the presence ofhSARS, or the polypeptide or mRNA or genomic RNA encoding thepolypeptide in the test sample.

The invention also encompasses kits for detecting the presence of hSARSor a polypeptide or nucleic acid of the invention in a test sample. Thekit, for example, can comprise a labeled compound or agent capable ofdetecting hSARS or the polypeptide or a nucleic acid molecule encodingthe polypeptide in a test sample and, in certain embodiments, a meansfor determining the amount of the polypeptide or mRNA in the sample(e.g., an antibody which binds the polypeptide or an oligonucleotideprobe which binds to DNA or mRNA encoding the polypeptide). Kits canalso include instructions for use.

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) which binds to apolypeptide of the invention or hSARS epitope; and, optionally, (2) asecond, different antibody which binds to either the polypeptide or thefirst antibody and is conjugated to a detectable agent.

For oligonucleotide-based kits, the kit can comprise, for example: (1)an oligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding a polypeptide of theinvention or to a sequence within the hSARS genome or (2) a pair ofprimers useful for amplifying a nucleic acid molecule containing anhSARS sequence. The kit can also comprise, e.g., a buffering agent, apreservative, or a protein stabilizing agent. The kit can also comprisecomponents necessary for detecting the detectable agent (e.g., an enzymeor a substrate). The kit can also contain a control sample or a seriesof control samples which can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container and all of the various containers are within asingle package along with instructions for use.

5.10 Screening Assays to Identify Anti-Viral Agents

The invention provides methods for the identification of a compound thatinhibits the ability of hSARS virus to infect a host or a host cell. Incertain embodiments, the invention provides methods for theidentification of a compound that reduces the ability of hSARS virus toreplicate in a host or a host cell. Any technique well-known to theskilled artisan can be used to screen for a compound that would abolishor reduce the ability of hSARS virus to infect a host and/or toreplicate in a host or a host cell.

In certain embodiments, the invention provides methods for theidentification of a compound that inhibits the ability of hSARS virus toreplicate in a mammal or a mammalian cell. More specifically, theinvention provides methods for the identification of a compound thatinhibits the ability of hSARS virus to infect a mammal or a mammaliancell. In certain embodiments, the invention provides methods for theidentification of a compound that inhibits the ability of hSARS virus toreplicate in a mammalian cell. In a specific embodiment, the mammaliancell is a human cell.

In another embodiment, a cell is contacted with a test compound andinfected with the hSARS virus. In certain embodiments, a control cultureis infected with the hSARS virus in the absence of a test compound. Thecell can be contacted with a test compound before, concurrently with, orsubsequent to the infection with the hSARS virus. In a specificembodiment, the cell is a mammalian cell. In an even more specificembodiment, the cell is a human cell. In certain embodiments, the cellis incubated with the test compound for at least 1 minute, at least 5minutes at least 15 minutes, at least 30 minutes, at least 1 hour, atleast 2 hours, at least 5 hours, at least 12 hours, or at least 1 day.The titer of the virus can be measured at any time during the assay. Incertain embodiments, a time course of viral growth in the culture isdetermined. If the viral growth is inhibited or reduced in the presenceof the test compound, the test compound is identified as being effectivein inhibiting or reducing the growth or infection of the hSARS virus. Ina specific embodiment, the compound that inhibits or reduces the growthof the hSARS virus is tested for its ability to inhibit or reduce thegrowth rate of other viruses to test its specificity for the hSARSvirus.

In one embodiment, a test compound is administered to a model animal andthe model animal is infected with the hSARS virus. In certainembodiments, a control model animal is infected with the hSARS viruswithout the administration of a test compound. The test compound can beadministered before, concurrently with, or subsequent to the infectionwith the hSARS virus. In a specific embodiment, the model animal is amammal. In an even more specific embodiment, the model animal can be,but is not limited to, a cotton rat, a mouse, or a monkey. The titer ofthe virus in the model animal can be measured at any time during theassay. In certain embodiments, a time course of viral growth in theculture is determined. If the viral growth is inhibited or reduced inthe presence of the test compound, the test compound is identified asbeing effective in inhibiting or reducing the growth or infection of thehSARS virus. In a specific embodiment, the compound that inhibits orreduces the growth of the hSARS in the model animal is tested for itsability to inhibit or reduce the growth rate of other viruses to testits specificity for the hSARS virus.

6. EXAMPLES

The following examples illustrate the use of siRNAs for preventing ortreating SARS infection. These examples should not be construed aslimiting.

6.1 Materials and Methods

siRNA

(1) siRNAs targeting the replicase 1A region of SCoV.

Because of the conserved nature of 5′-sequence, we designed six 21- and22-mer siRNAs targeting different sites of the replicase 1A region(FIGS. 1A and 1B). Double stranded siRNAs were synthesized by GENSET SALtd. (Paris, France). The sense strands of SARSi-1 (SEQ ID NO:1),SARSi-2 (SEQ ID NO:2), SARSi-3 (SEQ ID NO:3), SARSi-4 (SEQ ID NO:4),SARSi-5 (SEQ ID NO:5) and SARSi-6 (SEQ ID NO:6) correspond to thecoronavirus nucleotide sequences, 512 to 531, 586 to 604, 916 to 934,1194 to 1213, 3028 to 3046 and 5024 to 5042, respectively.

(2) siRNAs targeting the structural genes of SCoV.

Five 21-, 22- and 23-mer siRNAs targeting one site of each of the Sglycoprotein gene, E protein gene and N protein gene and two sites ofthe M protein gene of SCoV, respectively (FIGS. 4A and 4B), wereprepared according to the method described above. The sense strands ofSARSi-7 (SEQ ID NO:7), SARSi-8 (SEQ ID NO:8), SARSi-9 (SEQ ID NO:9),SARSi-10 (SEQ ID NO:10) and SARSi-11 (SEQ ID NO:11) correspond to thecoronavirus nucleotide sequences, 23165 to 23184, 26128 to 26148, 28663to 28682, 26652 to 26671 and 26575 to 26595, respectively.

In addition, GL2i (5′-CGUACGCGGAAUACUUCGATT-3′; SEQ ID NO:12), a siRNAtargeting luciferase mRNA (Elbashir, S M, et al. Duplexes of21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature 2001; 411: 494-498), was used as unrelated siRNA control. In someexperiments, SARSi-4, which showed the most potent siRNA targeting repas described above, was used as positive control.

Cell Culture, Transfection and Viral Infection

Unlike other human coronaviruses, it is possible to grow theSARS-associated coronavirus in monkey cells (Peiris, J S, et al., 2003,Coronavirus as a possible cause of severe acute respiratory syndrome,Lancet 361: 1319-1325). The anti-SARS activities of the six siRNA weretested in monkey kidney cells (FRhk-4 cells).

In general, about 3000 cells of FRhk-4 which had been cultured in MEMmedium with 10% FBS were seeded in each of 96 well and transfected with200 nM siRNA using Oligofectamine (Invitrogen, MD) according to themanufacturer's instructions. Four hours post-transfection, 10% fetalbovine serum (FBS) was added to the culture medium. After incubation foradditional four hours (total 8-hour incubation for SARSi1-6 and 6-hourincubation for SARSi7-12, post-transfection), the medium was removed,and the cells were washed once with PBS, and infected with one isolateof SARS-associated coronavirus (GZ50 strain) in PBS buffer atmultiplicity of infection (MOI) 0.05 for one hour. The cells were thenwashed twice with MEM medium and cultured in the same medium containing1% FBS for 36 hours for SARSi1-6 and 24 hours for SARSi7-12. ForSARSi1-6, pictures were taken before and after immunostaining (FIG. 2)using phase-contrast microscopy and fluorescent microscopy,respectively. Cytopathic effects (CPE) of the infected cells with orwithout SARSi7-12 were also recorded using phase-contrast microscopy(FIG. 5).

To test the effects of SARSi-4 on the replication of differentcoronavirus strains, additional three (3) strains (GZ34 strain isolatedfrom SARS patient in Guandong Province; and HKR1 and HKR2 strainsisolated in Hong Kong, respectively) were purified and used to infectFRhk-4 cells (see FIG. 3B).

Immunostaining

After infection with coronavirus for 36 hours, the cells were fixed with−20° C. ethanol for 10 min and coronavirus antigens were detected byindirect immunoflorescence assay (IFA) as described by Peiris et al(Coronavirus as a possible cause of severe acute respiratory syndrome.Lancet 2003; 361: 1319-1325). Briefly, after the cells were fixed withethanol, anti-SARS sera from SARS patients were added and incubated for30 min at room temperature. The cells were then washed with PBS 4 times,5 min each, and stained with FITC-labeled secondary antibody (St. Cruz,Calif. USA).

Quantitative Real-time PCR

The total RNA was isolated 36 hours after infection andreverse-transcription was applied. Real-time PCR was performed asdescribed previously (Poon, LLM, et al., 2003, Rapid Diagnosis of aCoronavirus Associated with Severe Acute Respiratory syndrome (SARS).Clin. Chem. 49(7): 1-3). Briefly, the cells in each well (about 3,000cells) were washed twice with PBS, and total RNA was extracted usingRNeasy Mini Kit (Qiagen, Germany) in accordance with the manufacturer'sinstructions. Reverse-transcription was performed using random hexamerswith the ThermoScript™ RT system (Invitrogen, Calif.).

Intracellular viral RNA was quantified using quantitative RT-PCR eitherby method A (see FIG. 3A) or B (FIGS. 6A and 6C). In method A, FastStartDNA Master SYBR Green I fluorescence reaction (Roche, Ind.) was used inthe PCR assay. Briefly, 2 μl of cDNA was amplified in 20 μl containing,per liter, 3.5 mmol of MgCl₂, 0.25 μmol of forward primer (coro3:5′-TACACACCTCAGCGTTG-3′; SEQ ID NO:13), and 025 μmol of reverse primer(coro4: 5′-CACGAACGTGACGAAT-3′; SEQ ID NO:14). Reactions were performedin a LightCycler® (Roche) with the following conditions: 10 min at 95°C.; followed by 5-cycles of 95° C. for 10 sec; and 72° C. for 9 sec.Plasmids containing the target sequence were used as positive controls.Fluorescence signals from these reactions were captured at the end ofthe extension step in each cycle. To determine the specificity of theassay, PCR products were subjected to melting curve analysis at the endof the assay (65° to 95° C.; 0.1° C./sec) (data not shown).

In method B, the forward primer (5′-GCTTAGGCCCTTTGAGAGAGACA-3′; SEQ IDNO:15), the reverse primer (5′-GCCAATGCCAGTAGTGGTGTAAA-3′; SEQ ID NO:16)and the fluorescent probe [5′-(TET®)CTAATGTGCCTTTCTCCCCTGATGGCA(TAMRA®)-3′; SEQ ID NO:17] which hybridized to the 5′-region of S gene,was used for real-time PCR (TaqMan® technology). Forward and reverseprimers (final concentration 900 nM), the fluorescent probe (finalconcentration 250 nM) and 2 μl of the RT product (template) were mixedwith Master Mix (Applied Biosystems, USA). The real-time quantificationwas carried out using ABI PRISM®7900 HT Sequence Detection System. PCRconditions employed were: 50° C. for 5 min; 95° C. for 10 min; then 40cycles of 95° C. for 15 sec; and 61° C. for 1 min.

In some experiments, the total intracellular RNA from infected cellswere isolated at different time points and quantified for viral genomicRNA copies (He, M. L., 2003, Jama 290:2665-2666; which is incorporatedherein by reference in its entirety) (see FIG. 6A).

To test whether the anti-viral activities of the siRNAs aredose-dependent, FRhk-4 cells were transfected with different amounts ofthe siRNAs, respectively. To normalize the transfection efficiency, GL2iwas used as a carrier and the final concentration of siRNAs (SARSi+GL2i)was maintained as 200 nM in the culture media (see FIG. 6C).

Back Titration of Virus In the Culture Media

The effects of siRNAs on viral titers were determined by back titrationexperiments in the culture media 24-hour post-infection. Viral titer isa parameter of live viruses, which reflects the actual viral genomereplication, packaging and secretion. Briefly, viral particles releasedinto the culture medium were quantified using a CPE-based TCID₅₀ test.Culture supernatant collected from SARS-CoV-infected cells 24 hoursafter viral infection was serially diluted at 10-fold with 1%-MEM andinoculated into FRhK-4-cells in 96-well plates. Results were evaluatedafter 3 days of culture under phase-contrast microscopy, and viraltiters were calculated (FIG. 6B).

Synergistic Effects by Combinations of siRNAs

To further test the antiviral activities at low dosage and possiblesynergistic effects, the inhibitory effect of the siRNA at 10 nM wasfirst studied (the left half of FIG. 6D). In some experiments, thecombinations of two different siRNAs (i.e., SARSi-4/7; SARSi-4/8;SARSi-4/9; SARSi-7/8; and SARSi-7/9) at equal amount, keeping the totalsiRNA concentration at 10 nM in the culture media (i.e., 5 nM each oftwo siRNAs), were tested by back titration (see the right half of FIG.6D).

6.2 Results and Discussion

FRhk-4 cells infected with coronavirus with or without GL2i exhibited asignificant morphological change with cytopathic effect (CPE, FIGS. 2-B,5-II and 5-III) in comparison with the uninfected negative control(FIGS. 2-A and 5-I). Uninfected cells were flattened, whereas theinfected cells became refractile and rounded up, and were floating awayor dead. No toxicity or CPE was observed when cells were transfectedwith siRNA alone (data, except for GL2i, not shown; for GL2i, see FIG.5-III). Transfection with SARSi-2, SARSi-3 and SARSi-4 markedlyinhibited the CPE caused by viral infection and replication (FIGS. 2-D,2-E and 2-F, respectively), whereas SARSi-1, SARSi-5 and SARSi-6 wereless effective (FIGS. 2-C, 2-G and 2-H, respectively), judged bymorphological changes. The cells transfected with SARSi-7 through 11were also protected from CPE (FIGS. 5-VI through 5-X, respectively),demonstrating that siRNAs that target structural genes also exhibitanti-SARS activities.

The results were further confirmed by immunostaining with antibodyagainst coronavirus antigens (FIGS. 2-I through 2-P). These findingsclearly demonstrated the inhibition of coronavirus infection andreplication by siRNAs (SARSi-2, 3, 4, 7, 8, 9, 10, and 11).

To determine the relative efficacy of their anti-coronavirus activities,the viral titers were determined by quantitative RT-PCR as describedpreviously (Poon et al., supra.). As shown in FIG. 3A, among siRNAstargeting the replicase 1A region, SARSi-4 was the most effective siRNA,against the coronavirus, which almost completely inhibited coronavirusinfection and replication, followed by SARSi-2 and SARSi-3. The viraltiter was reduced by 92% by SARi-4, 89% by SARSi-2 and 85% by SARSi-3,but by only 50% to 65% by the other three siRNAs. The siRNA specificallytargeting on luciferase mRNA (GL2i, see Elbashir, S M, et al. Duplexesof 21-nucleotide RNAs mediate RNA interference in cultured mammaliancells. Nature 2001; 411:494-498) did not exhibit anti-SARS activity(FIGS. 3A, 5-IV, 6A and 6B). SARSi-4 was also most effective inanti-SARS viral replication when compared with those targeting thestructural genes (i.e., SARSi7 through SARSi-11) in the study for thetime course of viral replication in the transfected cells (see FIG. 6A).The results on the inhibition of coronavirus replication in FIGS. 2 and3A as well as FIGS. 5 and 6B were consistent.

It is known that RNA viruses including coronavirus are usuallyassociated with rapid evolution and frequent mutations afterinterspecies transmission (Domingo, E, et al. RNA virus mutations andfitness for survival. Annu Rev Microbiol. 1997; 51:151-178).

We have isolated and purified four different stains of coronavirus,i.e., GZ34 stain, GZ50 stain, HKR1 stain and HKR2 stain, from SARSpatients in Guangdong Province and Hong Kong. The effects of SARSi-4 onthe replication of different coronavirus stains were examined using themethods as described, supra.

SARSi-4 markedly inhibited the replication of all three other strains(FIG. 3B). Therefore, the siRNAs targeting on the replicase gene will bethe best choice for the development of a broad range of anti-SARS drugs.Based on the present studies, SARSi-4 offers promise for developmentinto a new anti-SARS drug. Since the siRNA targets on the conserved RNAsequence of replicase region, SARSi-4 can be used for the treatment ofdifferent subtypes of coronavirus infections.

Since the use of combination drugs often exhibits synergistic effects,thereby allowing the lower dosages, the same possibility was explored bycombining siRNAs that target different gene sites. The inhibitory effectof individual siRNAs (SARSi-4, 7, 8, 9 and 10) used alone was lower at10 nM than at 200 nM. The viral titer was reduced 5-fold by SARSi-4,6-fold by SARSi-8, and 1-fold by SARSi-7, 9 and 10 without carrier siRNA(see the left half of FIG. 6D). No synergistic effects were observedwhen two or three effective siRNAs targeting rep gene were combined (He,M. L. et al., 2003, JAMA 290:2665-2666, which is incorporated herein byreference in its entirety). However, the viral titers were reduced18-fold by the combinations of SARSi4/7 and SARSi-7/8, respectively andbetween 6-fold and 12-fold by the combinations of SARSi-4/8, 4/9 and7/9, respectively. Thus, we demonstrated that siRNAs targetingfunctionally distinct genes exhibit synergistic effect even at the lowerdosages. The enhanced antiviral effects of siRNAs used in combination atthe low dosage indicate a high possibility of their use in clinicalapplications with high efficacy and reduced toxicity. As siRNA undergoestotally different metabolism from those of other types of antiviraldrugs, it may be used alone or in combination with other anti-viralagents, such as interferon-beta, to achieve more effective treatment forSARS.

Previous studies have reported an almost complete inhibition of HBVreplication by siRNAs/shRNAs (He et al., submitted). Other examples ofinhibition of gene expression may be found in Kapadia, S B, et al.Interference of hepatitis C virus RNA replication by short interferingRNAs. Proc. Natl. Acad. Sci. USA 2003; 100: 2014-2018; Randall, G, etal. Clearance of replicating hepatitis C virus replicon RNAs in cellculture by small interfering RNAs. Proc. Natl. Acad. Sci. USA 2003; 100:235-240; Wilson, J A, et al. RNA interference blocks gene expression andRNA synthesis from hepatitis C replicons propagated in human livercells. Proc. Natl. Acad. Sci. USA. 2003; 100: 2783-2788; Jacque, J M, etal. Modulation of HIV-1 replication by RNA interference. Nature 2002;418: 435-438; Lee, N S, et al. Expression of small interfering RNAstargeted against HIV-1 rev transcripts in human cells. Nat. Biotechnol.2002; 20: 500-505; and Novina, C D, et al. siRNA-directed inhibition ofHIV-1 infection. Nat. Med. 2002; 8: 681-686.

7. MARKET POTENTIAL

The recent worldwide outbreak of SARS has posed the urgent need foreffective vaccines and/or drugs for prevention and treatment of SARS.The siRNA disclosed herein are particularly useful for inhibiting SARSinfection and replication in humans and are good candidates for clinicaland research applications.

8. EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain manyequivalents to the specific embodiments of the invention describedherein using no more than routine experimentation. Such equivalents areintended to be encompassed by the following claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

1. An isolated nucleic acid molecule consisting of the nucleotidesequence of SEQ ID NO:2 and/or the complement thereof.
 2. An isolateddouble-stranded nucleic acid molecule, wherein the nucleotide sequenceof a first stand consists of the nucleotide sequence of SEQ ID NO:2 andthe nucleotide sequence of a second strand is complementary to thenucleotide sequence of the first strand.
 3. The nucleic acid molecule ofclaim 2, wherein said nucleic acid molecule is a RNA.